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ORIGINAL ARTICLE
Year : 2012  |  Volume : 8  |  Issue : 4  |  Page : 561-564

Norathyriol suppresses transformation in JB6 P+ cells by the inhibition of Akt


1 Department of Biochemistry, Guangdong Medical College, Dongguan, Guangdong, People's Republic of China, China
2 Sino-America Cancer Res Institute, Guangdong Medical College, Dongguan, Guangdong, People's Republic of China, China

Date of Web Publication29-Jan-2013

Correspondence Address:
Jixia Li
Department of Biochemistry, Guangdong Medical College, Dongguan, Guangdong 523808, People's Republic of China
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.106537

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 > Abstract 

Context: Chemoprevention has been acknowledged as an important and practical strategy for the management of skin cancer. Norathyriol, a naturally occurring compound present in various plants, has a potent anticancer-promoting activity.
Aims: The aim was to investigate the chemopreventive activity of norathyriol on JB6 P+ cells.
Materials and Methods: A soft agar assay was used to detect the effect of norathyriol on cell transformation. The activator protein-1 (AP-1) transactivation activity was examined by the luciferase assay.
Results: Norathyriol inhibited epidermal growth factor (EGF)- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation in a dose-dependent manner. The activation of activator protein-1 was dose dependently suppressed by norathyriol treatment. Western blot data revealed that norathyriol attenuated the phosphorylation of Akt.
Conclusions: Norathyriol exerts a potent chemopreventive activity by inhibiting Akt activation in neoplastic cell transformation.

Keywords: Carcinogenesis, chemoprevention, natural compound, norathyriol


How to cite this article:
Li J, Li X, He Z, Zhou K. Norathyriol suppresses transformation in JB6 P+ cells by the inhibition of Akt. J Can Res Ther 2012;8:561-4

How to cite this URL:
Li J, Li X, He Z, Zhou K. Norathyriol suppresses transformation in JB6 P+ cells by the inhibition of Akt. J Can Res Ther [serial online] 2012 [cited 2019 Sep 17];8:561-4. Available from: http://www.cancerjournal.net/text.asp?2012/8/4/561/106537


 > Introduction Top


The neoplastic transformation of cells is one of the major events occurring during carcinogenic processes. Activator protein (AP-1) acts as a pivotal transcription factor involved in the neoplastic transformation and development of cancer, [1],[2],[3],[4] and is regulated by upstream kinases, such as Akt signaling pathway. The Akt signaling pathway is critical for AP-1 activation in response to a wide variety of extracellular stimuli including 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF). The aberrant activation of Akt has been reported in various tumors including skin, ovarian, cervical, breast, prostate, and pancreatic cancer, [5],[6] and thus, the inhibition of Akt activation might be an effective method for intervening in skin carcinogenesis.

Chemoprevention has been acknowledged as an important and practical strategy for the management of cancer. Norathyriol (1, 3, 6, 7-tetrahydroxy-9H-xanthen-9-one), an aglycone of a xanthone C-glycoside mangiferin, is isolated from several plants including mango, Hypericum elegans, and Tripterospermum lanceolatum. Norathyriol is known to possess antioxidant, anti-inflammatory, and antitumor properties. [7],[8],[9] This compound has multiple targets and inhibits the transactivation of peroxisome proliferator-activated receptor isoforms, [10] ABCB1/P-glycoprotein activity, [7] protein kinase C activity, [9] cyclooxygenase and lipoxygenase activity, [8] and ERK activity. [11] It has been reported that norathyriol inhibits platelet aggregation, [12] relaxes the rat thoracic aorta, [13] and inhibits cutaneous plasma extravasation. [14] In addition, norathyriol is reported to induce Ca 2+ release from sarcoplasmic reticulum of the skeletal muscle, [15] and inhibit the PMA-induced neutrophil respiratory burst and aggregation. [16],[17] Hence, norathyriol could be a chemopreventive agent for skin cancer.

The JB6 mouse skin epidermal cell system, including promotion-sensitive (P+) and promotion-resistant (P−) components, allows the study of tumor promoter-induced carcinogenic processes at the molecular level. TPA and EGF induce large, tumorigenic, and anchorage-independent colonies in soft agar. [18] In this study, we examined norathyriol as a natural chemopreventive agent against skin cancer and its mechanism of antitumorigenic effects, using TPA and EGF as tumor promoters in the JB6 P+ mouse epidermal skin cell model. We report that norathyriol inhibits neoplastic cell transformation by the inhibition of Akt.


 > Materials and Methods Top


Norathyriol was obtained from Analyticon Discovery (Potsdam, Germany). Eagle's minimum essential medium (MEM), basal medium Eagle, was purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Gemini Bio-products (Calabasa, CA, USA). The antibodies against phosphorylated Akt (Ser 473) and total Akt were purchased from Cell Signaling Biotechnology (Beverly, MA, USA). The antibody against β-actin was purchased from Sigma-Aldrich. The protein assay kit was from Bio-Rad (Hercules, CA, USA) and the luciferase assay substrate was purchased from Promega (Madison, WI, USA).

The JB6 P+ cell line and JB6 cells stably transfected with an AP-1 luciferase reporter plasmid were cultured in monolayers at 37°C in a 5% CO 2 incubator in 5% FBS/MEM supplemented with penicillin/streptomycin (100 units/ml; Invitrogen).

JB6 P+ cells (8 × 10 3 /ml) were exposed to TPA (20 ng/ml) or EGF (10 ng/ml) with or without norathyriol (0-25 μM) in 1 ml of 0.33% BME agar containing 10% FBS or in 3 ml of 0.5% BME agar containing 10% FBS. The cultures were maintained at 37°C in a 5% CO 2 incubator for 7 or 14 days, after which the cell colonies were counted under a microscope with the aid of the Image-Pro Plus software program (v.4; Media Cybernetics, Silver Spring, MD, USA). [19]

Confluent monolayers of JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid were trypsinized, and 4 × 10 4 viable cells suspended in 1 ml of 5% FBS/MEM were added to each well of a 24-well plate. Plates were incubated overnight at 37°C in a humidified atmosphere of 5% CO 2 . Cells were starved in the serum-free medium for another 24 h. The cells were then treated for 2 h with norathyriol (0-25 μM) and then exposed to 20 ng/ml TPA or 10 ng/ml EGF and then harvested after 24 h. After treatment, cells were disrupted with 100 μl of the lysis buffer (0.1 M potassium phosphate buffer, pH 7.8, 1% Triton X-100, 1 mM dithiothreitol, and 2 mM EDTA), and the luciferase activity was measured using a luminometer (Luminoskan Ascent; Thermo Electron Corp., Helsinki, Finland).

After the cells (1 × 10 6 ) were cultured in a 10-cm dish overnight, they were starved in the serum-free medium for another 24 h to eliminate the influence of FBS on the activation of mitogen-activated protein kinases. The cells were then treated with norathyriol (0-25 μM) for 2 h before they were exposed to 20 ng/ml TPA or 10 ng/ml EGF and then harvested after 30 or 15 min. The harvested cells were disrupted, and the supernatant fractions were boiled for 5 min. The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad) as described in the manufacturer's manual. Lysate protein (30-50 μg) was subjected to 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare). After blotting, the membrane was incubated with the specific primary antibody at 4°C overnight. Protein bands were visualized by the chemiluminescence detection kit (GE Healthcare) after hybridization with the AP-linked secondary antibody.

As necessary, data were expressed as means ± SE and significant differences were determined using one-way ANOVA. A probability value of P < 0.05 was used as the criterion for statistical significance. All analyses were performed using Statistical Analysis Software (SAS, Inc.).


 > Results Top


We examined the effect of norathryiol on the EGF- and TPA-induced neoplastic transformation of JB6 P+ cells. Based on the numbers of transformed cell colonies, treatment with norathyriol significantly inhibited EGF- and TPA-promoted neoplastic transformation in a dose-dependent manner [Figure 1] and [Figure 2]. Norathyriol at 25 μM suppressed EGF- and TPA-induced cell transformation by 70% and 45%, respectively.
Figure 1: Norathyriol inhibits the EGF-induced cell transformation of JB6 P+ cells. Cells were treated as described under "Materials and methods" and colonies were counted under a microscope with the aid of Image- Pro Plus software (v.4). Data are shown as means ± SE. The asterisks (**) indicate a signifi cant difference (P < 0.001) between groups treated with EGF and norathyriol compared to the group treated with EGF alone

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Figure 2: Norathyriol inhibits the TPA-induced cell transformation of JB6 P+ cells. Cells were treated as described under "Materials and methods" and colonies were counted under a microscope with the aid of Image- Pro Plus software (v.4). Data are shown as means ± SE. The asterisks (**) indicate a significant difference (P < 0.001) between groups treated with TPA and norathyriol compared to the group treated with TPA alone

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To determine the molecular mechanism of norathyriol's cancer chemopreventive activity, we measured the effect of norathyriol on the transactivation of AP-1 using JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid. Norathyriol attenuated EGF- and TPA-induced transactivation of AP-1 [Figure 3] in a dose-independent manner, which may contribute to its antitumor-promoting activities.
Figure 3: Norathyriol blocks the EGF/TPA-induced AP-1 transactivation in JB6 cells. For the luciferase assay, JB6 cells stably transfected with an AP-1 luciferase reporter plasmid were cultured in 5% FBS/MEM. Cells were starved in the serum-free medium for 24 h, and then treated with norathyriol (0– 25 μM), or its vehicle DMSO (control), in 5% FBS/ MEM for 2 h. Cells were then exposed to 10 ng/ml EGF (upper panel) or 20 ng/ml TPA (lower panel) and harvested at 12 h. The luciferase activity was assessed and the AP-1 activity was expressed relative to control cells without EGF or TPA treatment. Data are shown as means ± SE. The asterisk (*) indicates a signifi cant difference (P < 0.05) between groups treated with EGF/TPA and norathyriol compared to the group treated with EGF/TPA alone

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AP-1 activation is regulated by upstream kinases, such as Akt signaling. Hence, we examined the effect of norathyriol on Akt activation. We found that norathyriol strongly suppressed the EGF- and TPA-induced phosphorylation of Akt [Figure 4]. These results suggested that the inhibition of Akt activation by norathyriol leads to the suppression of AP-1 transactivation, resulting in antitumor effects.
Figure 4: Norathyriol inhibits the EGF/TPA-induced phosphorylation of Akt. Cells were treated with norathyriol at the indicated concentrations (0– 25 μM) for 2 h and then exposed to 10 ng/ml EGF or 20 ng/ml TPA and harvested 15 or 30 min later. The levels of phosphorylated and total Akt proteins were determined by western blot analysis. Semiquantitative analysis was performed by Image J software

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 > Discussion Top


The use of chemopreventive agents (especially naturally occurring plant products) to inhibit skin carcinogenesis, particularly at the promotion stage, is gaining attention. Norathyriol is the main metabolite of mangiferin in vivo, deriving from a deglycosilation process. [20],[21] Norathyriol possesses antioxidant, anti-inflammatory, and antitumor effects. Recent studies showed that norahtyriol is more active than mangiferin. [7],[10] In the present study, we have shown norathyriol against EGF- and TPA-induced skin carcinogenesis and identified the molecular mechanism(s).

The neoplastic transformation of cells is considered to be a major event occurring during carcinogenesis. [22] The inhibition of transformation is a key preventive measure. Our result showed that norathyriol (25 μM) significantly inhibited EGF- and TPA-promoted cell transformation in JB6 P+ cells. This study suggested that the inhibition of cell transformation might be an important mechanism explaining the anticarcinogenic effects of norathyriol.

AP-1 is extremely important in the processes of cell proliferation, differentiation, inflammation, survival, and transformation. [1],[2],[3],[4],[23] Therefore, AP-1 plays an important role in tumorigenesis. Moreover, recent studies have revealed a role of Akt in the activation of AP-1. [24] AP-1 and Akt are key molecules activated after EGF or TPA exposure. Therefore, we performed a luciferase assay to detect the AP-1 transcription activity and western blotting to assess Akt phosphorylation. Our results showed that norathyriol inhibits AP-1 transcription activation through the suppression of Akt phosphorylation.


 > Conclusion Top


Norathyriol inhibits skin cell carcinogenesis. This inhibition occurs mainly through the suppression of transformation and the regulation of AP-1 by inhibiting Akt activation.


 > Acknowledgment Top


The authors are grateful to Guangdong Medical College for the research grant (no. XG-1101 and GX0306) and Guangdong Natural Science Foundation (S2012010008259).

 
 > References Top

1.Dong Z, Birrer MJ, Watts RG, Matrisian LM, Colburn NH. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc Natl Acad Sci U S A 1994;91:609-13.  Back to cited text no. 1
    
2.Huang C, Ma WY, Young MR, Colburn N, Dong Z. Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells. Proc Natl Acad Sci U S A 1998;95:156-61.  Back to cited text no. 2
    
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10.Wilkinson AS, Monteith GR, Shaw PN, Lin CN, Gidley MJ, Roberts-Thomson SJ. Effects of the mango components mangiferin and quercetin and the putative mangiferin metabolite norathyriol on the transactivation of peroxisome proliferator-activated receptor isoforms. J Agric Food Chem 2008;56:3037-42.  Back to cited text no. 10
    
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16.Hsu MF, Raung SL, Tsao LT, Lin CN, Wang JP. Examination of the inhibitory effect of norathyriol in formylmethionyl-leucyl-phenylalanine-induced respiratory burst in rat neutrophils. Free Radic Biol Med 1997;23:1035-45.  Back to cited text no. 16
    
17.Wang JP, Raung SL, Tsao LT, Lin CN. Evidence for the involvement of protein kinase C inhibition by norathyriol in the reduction of phorbol ester-induced neutrophil superoxide anion generation and aggregation. Eur J Pharmacol 1997;336:81-8.  Back to cited text no. 17
    
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19.Colburn NH, Wendel EJ, Abruzzo G. Dissociation of mitogenesis and late-stage promotion of tumor cell phenotype by phorbol esters: Mitogen-resistant variants are sensitive to promotion. Proc Natl Acad Sci U S A 1981;78:6912-6.  Back to cited text no. 19
    
20.Bock C, Waldmann KH, Ternes W. Mangiferin and hesperidin metabolites are absorbed from the gastrointestinal tract of pigs after oral ingestion of a Cyclopia genistoides (honeybush tea) extract. Nutr Res 2008;28:879-91.  Back to cited text no. 20
    
21.Wang H, Ye G, Ma CH, Tang YH, Fan MS, Li ZX, et al. Identification and determination of four metabolites of mangiferin in rat urine. J Pharm Biomed Anal 2007;45:793-8.  Back to cited text no. 21
    
22.Kang NJ, Lee KW, Lee DE, Rogozin EA, Bode AM, Lee H J, et al. Cocoa procyanidins suppress transformation by inhibiting mitogen-activated protein kinase kinase. J Biol Chem 2008;283:20664-73.  Back to cited text no. 22
    
23.Cooper SJ, Bowden GT. Ultraviolet B regulation of transcription factor families: Roles of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) in UVB-induced skin carcinogenesis. Curr Cancer Drug Targets 2007;7:325-34.  Back to cited text no. 23
    
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